Pig sperm are highly susceptible to cryopreservation protocols; oxidative stress as well as drastic changes in plasma membrane fluidity alters sperm physiology. Use of pre-freeze cooling to sub-zero temperatures and antioxidants are experimental approaches directed to reduce the occurrence of sperm plasma membrane hyper-fluidity, and oxidative stress during cryopreservation. The objective of this work was to assess the effect of pre-freeze cooling to -5°C, and melatonin (MLT) as an antioxidant, on boar sperm plasma membrane fluidity and cryosurvival. After collection, ejaculates were diluted, transported (34 °C, 90 min), centrifuged and the supernatant removed. Only ejaculates showing at least 80% progressive motility and 85% viability of sperm were included in the experiments. In the first, sperm (3 boars, 9 ejaculates) were resuspended in lactose-egg yolk freezing medium (3% glycerol), and packaged in 0.5 mL plastic straws; in the second, sperm (4 boars, 18 ejaculates) were resuspended in BF5 freezing medium (1% glycerol) containing 0, 1 or 2 mM melatonin (MLT), and packaged in 0.25 mL plastic straws. Each ejaculate was split and all treatments were applied to aliquots of one ejaculate. Diluted sperm were cooled from 23 to 5 °C into a common refrigerator over 2 h. One half of the straws at 5 °C was frozen over nitrogen vapours and immersed in liquid nitrogen; the other half was further cooled to -5°C, and then frozen. An insulated box containing crushed saline ice (10% w/v) at −12°C was used to cool the straws to -5 °C . Straws were thawed in a water bath at 39 °C for 30 sec. Sperm were assessed for progressive motility (visual), viability (eosin-nigrosine), plasma membrane integrity (SYBR14/PI) and fluidity (MC540), acrosome integrity (PSA-FITC), and capacitation status (CTC assay). Data was analysed by “t” test and ANOVA. In the first experiment, there were no differences between cooling treatments for sperm quality; MC540 high-binding cells (hyper-fluid membranes) were 52, 57, and 11% for cooling to +5°C, -5°C, and before cooling respectively. In the second experiment, there were differences (P<0.05) between cooling treatments in plasma membrane integrity, non-capacitated acrosome-intact, and capacitated acrosome-intact spermatozoa (+5°C better than -5°C). There were no differences in sperm quality between MLT treatments. MC540 high-binding cells were 34, 32, and 32% for 0, 1 and 2 mM MLT, respectively. In conclusion, percentage of hyper-fluid membranes increased after freeze-thawing regardless of MLT and pre-freeze cooling temperature. Pre-freeze cooling was more important than MLT in boar sperm cryosurvival.
Supported by UNAM (PAPIIT IA204917, IA220419, PIAPI 1615/1649/1810).